Fish and other types of marine organisms lose their freshness very rapidly after death. Furthermore, the quality of canned salmon, tuna, crab and the like is largely dependent upon the freshness of the fish or shellfish used for processing. Freshness of fish can rarely be visually determined because it is often sold in frozen or processed form.
From the standpoint of consumer protection and food hygiene, extensive research has been focused on the development of reliable and inexpensive methods of determination of fish freshness. This is urgently required in food industries since fish freshness is an important factor in the preparation of high-quality products. Indicators of fish freshness such as ammonia, amines, volatile acids, catalase activity, trimethylamine (TMA) and nucleotides have so far been proposed. Among these chemicals, nucleotides produced by adenosine triphosphate (ATP) decomposition are considered the most reliable and useful indicators. In recent years, considerable attention has been focused on nucleotide degradation in fish muscle as a reliable indicator of the freshness of raw fish.
Immediately after death, ATP in fish muscles is autolytically degraded to hypoxanthine/xanthine through the following autolytic pathway: EQU ATP.fwdarw.ADP.fwdarw.AMP.fwdarw.IMP.fwdarw.HxR.fwdarw.Hx.fwdarw.X(1)
wherein
ATP is adenosine triphosphate PA1 ADP is adenosine diphosphate PA1 AMP is adenosine monophosphate PA1 IMP is inosine monophosphate PA1 HxR is inosine PA1 Hx is hypoxanthine PA1 X is xanthine.
Whereas IMP is one of the major contributing factors to the pleasant flavor of fresh fish, the accumulation of Hx and/or X during the storage results in an "off-taste". Several researchers have recognized that simultaneous determination of each nucleotide is necessary for a rapid estimation of freshness. From these observations, the concept of the K value was developed, in which: ##EQU1##
In several fish species, however, ATP and ADP concentrations rapidly decrease and are negligible within 24 hours after death. Similarly, a rapid decline of AMP is also observed and its concentration is somewhat less than 1 .mu.mol/g. In contrast to such behavior, IMP increases in the period ranging between 5 and 25 hours after death and then gradually decreases while the concentrations of HxR and Hx increase proportionally. In practice, the first measurements of fish freshness are usually performed at least 24 hours after death, thereby simplifying the determination of the K value in the following manner: ##EQU2##
A low K value should be expected for fresh fish. It is generally believed that fish having a K value of less than 0.2 has excellent freshness qualities while fish exhibiting a K value ranging between 0.2 and 0.4 has good freshness qualities. The increase in the rate of the K value depends on the type of fish since changes in the K value are based on the enzymatic reactions within the fish meat. The K value also varies appreciably with temperature even among the same fish species.
Based on these facts, various freshness determination methods have been developed. For example, Uchiyama et al. (Bulletin of the Japanese Society of Scientific Fisheries, Vol. 36, 977 (1970)) made an analysis of the various nucleotides found in fish muscle by using liquid chromatography to show that a deterioration in freshness can be detected from an increase in the K value. ##EQU3##
It was later determined by Nunata et al. in Journal of Japanese Society of Food Science and Technology, Vol. 28, 542 (1981) and by Kitada et al. in Journal of Japanese Society of Food Science and Technology, Vol. 30, No. 3, 151-154 (1983), that this method could also be used to determine the degree of freshness of poultry such as chicken.
However, the Uchiyama method has serious drawbacks, namely the necessity to use expensive liquid chromatography equipment that must be operated by skilled technicians, the time consuming separation and column regeneration as well as the difficulty in separating inosine from hypoxanthine.
Fujii et al. (Bulletin of the Japanese Society of Scientific Fisheries, Vol. 39, 69-84 (1973)) developed a method to estimate fish freshness based on the determination of the concentrations of IMP, HxR and Hx through enzymatic reactions. This method is based on the following equations: ##EQU4##
The IMP ratio has a high value when the degree of freshness is high and decreases as the degree of freshness decreases. For example, canned tuna having an IMP ratio of 40% or higher can be judged as having been processed from raw tuna having a high degree of freshness.
Unfortunately, this method also presents serious drawbacks. Hence, an expensive ultraviolet spectrophotometer must be used to conduct certain measurements and two expensive enzymes are necessary in order to conduct a blank measurement and this enzymatic reaction is time consuming. Furthermore, corrosive perchloric acid must be used as the extractant since the ultraviolet absorbing properties of trichloroacetic acid render the latter unsuitable for use as the extractant.
The determination of the K value by monitoring oxygen consumption using a Clark oxygen electrode has been commercially exploited by Oriental Electric Co. Ltd. The apparatus is known as the KV-101 freshness meter (hereinafter referred to as the K-meter) and comprises a Clark oxygen electrode attached to a reaction chamber.
A major drawback of this technique is the low sensitivity and the requirement of a rigid control of pH and oxygen tension. In addition, the K-meter uses soluble enzymes which cannot be reused and there is a gradual loss of the probe's sensitivity, presumably due to the fowling of the electrode by the enzymes and/or compounds in fish extract. Other techniques of fish freshness determination have also been developed in recent years.
Karube et al. (J. Agric. Food Chem. 32, 314-319, 1984) described an enzyme sensor system for the determination of the K value. The system combined a double membrane consisting of a 5'-nucleotidase membrane and a nucleoside phosphorylase-xanthine oxidase membrane with an oxygen electrode. A small anion-exchange resin column was also connected with the enzyme sensor for separation of nucleotides. This biosensor system is less desirable for practical application since the three compounds in the mixture (IMP, HxR and Hx) had to be separated by an elaborate scheme using four different buffers and an anion exchange column, amounting to a very complicated procedure.
Ohashi et al. (U.S. Pat. No. 4,650,752) disclosed an enzymatic method for determining the K-value for fish and molluscs using soluble enzymes together with an oxygen electrode. The enzymes used for determining inosine and hypoxanthine concentrations are nucleoside phosphorylase and xanthine oxidase. To determine the concentration of the decomposition products of adenosine triphosphate, the enzymes alkaline phosphatase, adenylic acid kinase, AMP deaminase and adenosine deaminase in a crude extract obtained from calf intestine as well as nucleoside phosphorylase and xanthine oxidase are used. Again, the main drawback of this technique is the low sensitivity, the requirement of several enzymes and the costly enzymes that cannot be reused. Although Ohashi et al. state that the measurement can be determined electrochemically from the amount of hydrogen peroxide produced, no experimental data were presented to substantiate this statement.
Therefore, an inexpensive and rapid method useful in monitoring fish freshness would be highly desirable.